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Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous... 4826–4833 Nucleic Acids Research, 2001, Vol. 29, No. 23 © 2001 Oxford University Press Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells Palaniyandi Manivasakam, Jiri Aubrecht, Samy Sidhom and Robert H. Schiestl* Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA Received August 8, 2001; Revised and Accepted October 18, 2001 ABSTRACT which may be lethal. Inappropriate repair of DSBs can result in chromosomal rearrangements, which can lead to cell Mammalian cells repair DNA double-strand breaks by transformation. illegitimate end-joining or by homologous recombin- Saccharomyces cerevisiae cells repair DSBs by homologous ation. We investigated the effects of restriction recombination, mainly mediated by gene products in the enzymes on illegitimate and homologous DNA RAD52 pathway. In the absence of RAD52, yeast cells repair integration in mammalian cells. A plasmid containing DSBs by non-homologous end joining (NHEJ), which requires the neo expression cassette, which confers G418 the Ku homologous proteins. NHEJ has been studied in yeast amongst other approaches by transformation with a linear resistance, was used to select for illegitimate integra- DNA molecule that contains a selectable marker, which does tion events in CHO wild-type and xrcc5 mutant cells. not have any homology to the genome. Schiestl and Petes (1) Co-transfection with the restriction enzymes BamHI, used a BamHI fragment containing the URA3 marker to trans- BglII, EcoRI and KpnI increased the efficiency of form a yeast strain in which the entire URA3 gene had been linearized plasmid integration up to 5-fold in CHO deleted. After addition of the BamHI restriction enzyme to the cells. In contrast, the restriction enzymes did not transformation mixture the efficiency of integration increased increase the integration efficiency in xrcc5 mutant several-fold and integrations occurred into BamHI sites. These cells. Effects of restriction enzymes on illegitimate events were designated restriction enzyme-mediated integra- and homologous integration were also studied in tion (REMI) events. This study was extended to investigate mouse embryonic stem (ES) cells using a plasmid how two different compatible and non-compatible ends are containing the neo gene flanked by exon 3 of Hprt. repaired (2), by restricting the DNA with one enzyme and adding another enzyme to the transformation mixture. The enzymes BamHI, BglII and EcoRI increased the Mammalian cells repair DSBs both by homologous recombin- illegitimate integration efficiency of transforming ation and by illegitimate recombination (IR). However, DNA several-fold, similar to the results for CHO cells. homologous integration frequencies are 100–1000-fold less However, all three enzymes decreased the absolute frequent than illegitimate integration (3,4), the major obstacle frequency of homologous integration ∼2-fold, and for gene targeting in mammalian cells. The molecular mechanism the percentage of homologous integration decreased of recombination in mammalian cells is under intensive >10-fold. This suggests that random DNA breaks investigation. Different complementation groups of ionizing attract illegitimate recombination (IR) events that radiation-sensitive rodent cell mutants have been identified and compete with homology search. three of them designated X-ray repair cross-complementing (XRCC) groups 5, 6 and 7 (5). The genes defective in groups 5 and 7, deficient in the mutants xrcc5 and xrcc7, respectively, INTRODUCTION encode components of a DNA-dependent protein kinase In the eukaryotic genome, DNA double-strand breaks (DSBs) (DNA-PK), a complex possessing DNA end-binding and can occur during cellular processes such as DNA repair, protein kinase activity (6–8). The DNA-binding subunit of recombination and replication; the early prophase of meiosis, DNA-PK is a heterodimer of 70- and 80-kDa subunits, named V(D)J recombination or as the result of exposure to DNA Ku70 and Ku80, respectively. Ku80 is deficient in group damaging agents. The repair of DSBs is important for the 5 cells (9). Ku is an abundant nuclear protein identified originally maintenance of genomic integrity and cellular survival because as an autoantigen from various autoimmune patients (10). The unrepaired DSBs will result in the loss of genetic information, Ku protein binds to free double-stranded DNA ends with 5′-or *To whom correspondence should be addressed at present address: Departments of Pathology and Environmental Health, UCLA School of Medicine and Public Health, 650 Charles E. Young Drive South, 71-295 CHS, Los Angeles, CA 90095, USA. Tel: +1 310 267 2087; Fax: +1 310 267 2578; Email: rschiestl@mednet.ucla.edu Present addresses: Palaniyandi Manivasakam, CombinatoRx, 650 Albany Street, Boston, MA 02118, USA Jiri Aubrecht, Pfizer Global Research and Development, Groton, CT 06340, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors Nucleic Acids Research, 2001, Vol. 29, No. 23 4827 3′-single-stranded protruding (PSS) ends or blunt ends, nicked from the 5′ end) in plasmid J3N. Plasmid pcDNA3.1/His/lacZ DNA, and duplex DNA ending with stem–loop structures (10), (Invitrogen, Carlsbad, CA) control vector has been used for which suggests a possible role in DNA repair and recombination. transient transfection to determine the effect of restriction These mutants are defective in DSB rejoining and have been enzymes on DNA uptake. shown to possess defects in components of DNA-PK. They Cell lines also exhibit defects in the rejoining steps in V(D)J recombination, a site-specific recombination process required for rearrangement CHO cells (CHO-K1; ATCC) and the xrs5 mutant cells of DNA to generate variability in genes encoding immuno- (obtained from Penny Jeggo, MRC Cell Mutation Unit, globulins. University of Sussex, UK) were grown in Cellgro DMEM Several investigators have utilized restriction enzymes to (Mediatech, Herndon, VA) supplemented with proline (3.7 mM), study the effect of DSB repair in mammalian cells. It has been 10% fetal bovine serum (Life Technologies, Rockville, MD), shown that introduction of restriction enzymes into mamma- 100 U/ml penicillin and 100 µ g/ml streptomycin in a humidified lian cells, either by electroporation or by calcium precipitation, air incubator at 37°C, 5% CO and 95% humidity. Embryonic produces cell killing, chromosomal aberrations, gene amplifi- stem (ES) cells (ES-D3; ATTC) were cultivated on monolayers of cation and increased mutation frequency at several loci [for irradiated feeder cells (STO-TN) in DMEM supplemented with review (11)]. Co-transfection with restriction enzymes HindIII 10% fetal bovine serum (Life Technologies), LIF (100 U/ml), and XbaI induces DNA integration into mouse Ltk cells (12). 0.2 mM mercaptoethanol (Life Technologies), 100 U/ml penicillin Inducible expression of EcoRI in CHO cells showed toxicity and 100 µ g/ml streptomycin (Mediatech) in a humidified air and chromosomal aberrations (13). incubator at 37°C, 5% CO and 95% humidity. The feeder cell line was developed from embryonic fibroblast cells (STO; Investigators showed that intrachromosomal homologous ATTC). That feeder cell line was transfected with plasmid PMA159 recombination can be induced by electroporation with restric- and selected in the presence of G418 (Life Technologies). One tion enzymes or intracellular expression of restriction enzymes stable transfected clone of STO fibroblast cells (STO-TN) (14,15). Using a loss of function assay in the APRT gene, conferring resistance to 1 mg/ml G418 was selected for prepara- Sargent et al. (16) showed that DSBs created by I–SceI tion of feeder cells. The STO-TN cell line carries a mutated induced homologous recombination 100-fold between repeats, hprt gene and neo expression cassette that allows simulta- but IR was stimulated 1000-fold. This indicates that the prefer- neous selection with 6-thioguanine (6-TG) and G418. A 90% ential mode of DSB repair in mammalian cells is NHEJ. confluent culture of STO cells was trypsinized, suspended in In the present study, we investigated the effects of restriction DMEM and irradiated with a 50 Gy dose of γ irradiation with a enzymes on illegitimate integration and homologous gene Co γ-ray source at a dose rate of 12.2 cGY/s. After irradiation, targeting in mammalian cells. We found that restriction the cells were frozen until further use. The frozen feeder cells were enzymes increased the frequency of illegitimate integration, thawed 3–10 days before seeding of ES cells, then plated at a which was dependent on the Ku80 function. Surprisingly, we density of 2.5–4 × 10 cells per 10 cm dish and grown to near also found that the restriction enzymes decreased the confluency. frequency of homologous integration, indicating a direct competition between the two pathways. Electroporation of cells CHO or xrs5 cells were grown to 90% confluence in 225 cm MATERIALS AND METHODS culture flasks (Corning, Corning, NY) and trypsinized using trypsin/EDTA (Mediatech). The detached cells were collected Plasmids in 10 ml of fresh culture medium, washed twice with phosphate- Plasmid PMA159 was derived from pUC and contains the neo buffered saline (PBS) and resuspended in electroporation gene. This plasmid was constructed in two steps. Digestion of buffer (272 mM sucrose, 7 mM Na HPO ,1mM MgCl ,pH 7.4). 2 4 2 pBK-CMV plasmid (from Stratagene) with AvrII restriction Approximately 3–5 × 10 cells in 0.8 ml of electroporation enzyme liberated the 1.6 kb neo expression cassette. BamHI buffer were mixed with 15 µ g of linearized plasmid linkers were ligated to the ends of the fragment after filling in (PMA159), 30–480 U of restriction enzyme or an appropriate the 5′ single-stranded ends with Klenow. neo was inserted amount of restriction enzyme buffer and restriction enzyme into the BamHI site of pUC. After partial digestion, the 5′ end storage buffer. The electroporation was performed using Bio-Rad of the BamHI site was eliminated by filling in the single-stranded Gene Pulser at 0.3 kV, 960 µ Fand 200 Ω . The electroporated ends and ligating the blunt ends. Plasmid J3N contains the neo cells were diluted with fresh culture medium, plated on to three gene flanked by exon 3 of the Hprt gene. A 7 kb BamHI frag- 10 cm dishes (Falcon) and incubated. After 24 h, the medium ment containing exons 2 and 3 of the Hprt gene was ligated was supplemented with G418 to a final concentration of 750 µ M. into pUC19 missing an EcoRI site. The neo expression To determine the viability after electroporation, 100 and 500 cells cassette obtained by AvrII digestion was blunt ended with were plated on to two 10 cm dishes. To minimize the occurrence Klenow, and XhoI linker was attached. The neo fragment of satellite clones the cultures were incubated without disturbance containing XhoI ends was ligated into the XhoI site, which for 11–14 days. The colonies of surviving cells were stained residesinexon3ofthe Hprt gene, yielding plasmid J3N. This using Giemsa stain (Sigma, St Louis, MO). plasmidhas homology to Hprt of 4.5and 2.5kbflankingthe ES cells were growntoconfluenceon10cmdishes covered neo fragment. J3NB plasmid was constructed by filling in two with STO-TN feeder cells. After trypsinization the cells BglII sites (located 1 and 3.5 kb from the 5′ end), J3NR by were resuspended in fresh medium and washed twice in filling in the two EcoRI sites(located4and 5.5kbfrom the PBS. Approximately 8–12 × 10 cells were electroporated in 5′ end) andJ3NH byfilling inthe HindIII site (located 3.7 kb PBS using Bio-Rad Gene Pulser at 0.3 kV and 500 µ F. The 4828 Nucleic Acids Research, 2001, Vol. 29, No. 23 electroporated ES cells were seeded on three 10 cm dishes producing PSS ends (19), was used. Except for PvuII, which covered with STO-TN feeder cells. After 24 h of incubation, showed a 50% reduction in colony forming unit (CFU), none the medium was supplemented with 500 µ g/ml G418. The of the other enzymes showed any significant level of reduction surviving colonies of stable integrants including random and in CFU with 30 U (data not shown). At the highest concentra- homologous recombination events were counted after 10 days. tion of 480 U, the enzymes BglII and PvuII, but none of the At that time, the medium was replaced with one containing other enzymes used including PstI, decreased the CFU 30 µ M 6-thioguanine (TG) (Sigma). Surviving colonies were significantly (data not shown). These results indicate that the counted after 5–7 days. restriction enzymes that were used for the REMI experiment were not toxic to the mammalian cells at the concentration Transient transfection to determine DNA uptake used for integration studies (30 U). Plasmid pcDNA3.1/His/lacZ (Invitrogen) was digested with REMI events in yeast usually integrate into restriction sites BglII, and cells were prepared and electroporated with the in the genome by micro-homology-mediated integration, and linear plasmid as described above in the presence or absence of both restriction sites at the ends of the integrating DNA are the BglII enzyme. The control in the absence of the enzyme maintained (1,20). This facilitates detection of such an event contained an equivalent amount of the enzyme storage buffer. simply by digestion of the genomic DNA with the same restric- The cells were then incubated overnight in the presence of tion enzyme that was used for REMI, and performing a X-gal. Cells were counted and screened for blue color under Southern blot that reveals the fragment size of the integrating the microscope. vector. We performed Southern blot analysis with twelve clones obtained after a transformation with the BamHI- Southern blot analysis digested plasmid PMA159 in the presence of BamHI (data not Stable transformants were obtained by transforming the cells shown). This experiment indicates that a single copy integra- with PMA159 plasmid, which was restricted with BamHI and tion event recreated both flanking BamHI sites inonly1 out of transfected with or without BamHI enzyme or with the plasmid 12 transformed clones. In the control in which no enzyme was digested with KpnI and transfected with or without KpnI. added, none of the events was flanked by BamHI sites. In the Individual clones were expanded by growing in 10 cm dishes. experiment with KpnI, 1 out of 10 events recreated the flanking The genomic DNA was isolated and Southern blot analyses KpnI sites, and in the control, none of the events recreated both were performed according to standard procedures using the KpnI sites. Control cells as well as restriction enzyme-treated 1.6 kb neo gene as probe. cells showed multiple fragments of various sizes, suggesting multiple copies at a single site and/or multiple integration events at multiple sites. RESULTS It has been reported that radiosensitive cells are more susceptible to the toxic effects and chromosome aberrations We investigated the effects of restriction enzymes on illegiti- caused by restriction enzymes (21). We used the xrs5 cell line mate and homologous DNA integrations in mammalian cells. that is deficient in Ku80 to study the toxicity and integration- To study IR, a plasmid containing the neo selection marker mediating effect of restriction enzymes. Approximately 55% was used. A DNA fragment containing the neo gene was of the cells survived with 30 U of the BamHI enzyme, while cloned into the BamHI site of pUC plasmid and one BamHI only 14% survived with 60 U (Table 2). In the presence of 30 site was eliminated by filling in and religation. All multi- and60U of the BamHI enzyme, REMI efficiency decreased to cloning sites are unique in this plasmid. The plasmid was ∼25 and 8%, respectively, in xrs5 cells, which is a greater linearized with various restriction enzymes and transfected in decrease than caused by toxicity alone (Table 2). Thus, Ku80 the presence of enzyme into the cells by electroporation. seems to be required for REMI. Initially we determined the effects of different concentrations (30, 120 and 480 U per transfection vial) of the BamHI enzyme Next, we investigated the effects of restriction enzymes on on the frequency of integration. The same increase in the the efficiency of gene targeting in ES cells. For this study, we efficiency of integration was seen with all three concentrations, constructed a plasmid containing the neo gene as a selec- and thus, 30 U were used for all further experiments. Since tion marker flanked by the Hprt gene (Fig. 1). This plasmid restriction enzyme buffers can produce chromosomal aberrations can be used to select for illegitimate integration as well as (17,18), the same concentration of restriction enzyme buffer for gene targeting. Integration of the plasmid will give rise and storage buffer as used for the restriction digests was used to G418-resistant clones. Homologous integration results in in all controls. The restriction enzymes BamHI, BglII and disruption of the Hprt gene, which can be selected for with EcoRI, which produce 5′ PSS ends, and KpnI, which produces 6-TG. As the hprt locus is hemizygous in XY ES cells, 3′ PSS ends, increased the efficiency of illegitimate integration targeted clones can be isolated by direct selection in 6-TG 2.5–5-fold (Table 1) in CHO cells. PstI, SmaIand HindIII for lack of hprt function. We tested the effect of BamHI, restriction enzymes, producing 3′ PSS ends, blunt ends and BamHI-E77K, BglII, EcoRI and HindIII restriction enzymes 5′ PSS ends, respectively, did not induce REMI. on gene targeting in ES cells. In the presence of BamHI, BglII Addition of PstI decreased the integration efficiency 2-fold. and EcoRI the total number of integration events increased We wanted to determine whether this decrease was due to 2–4-fold as seen in CHO cells (Table 3). Surprisingly, possible toxic effects of the enzyme. The cells were transfected however, in the presence of enzyme, the actual number of with three different concentrations: 30, 120 and 480 U for each homologous integration events decreased >2-fold, and the of the enzymes used in this study. As a control, PvuII, which is percentage of homologous integration as a fraction of all known to be a more potent inducer of toxic effects and integration events decreased down to >10-fold (Table 3). The chromosomal aberrations in mammalian cells than enzymes addition of HindIII, on the other hand, did not increase the Nucleic Acids Research, 2001, Vol. 29, No. 23 4829 Table 1. Effect of different restriction enzymes on REMI in CHO cells Plasmid digested with Enzyme added Integration (fold over control) No. of clones Significance BamHI – 1 (298) BamHI BamHI 3.74 ± 0.28 (1009) ** BglII – 1 (177) BglII BglII 5.49 ± 0.49 (928) ** EcoRI – 1 (223) – EcoRI EcoRI 2.40 ± 0.24 (479) ** PstI – 1 (935) – PstI PstI0.46 ± 0.05 (420) ** SmaI – 1 (254) – SmaI SmaI0.84 ± 0.05 (233) – KpnI – 1 (445) – KpnI KpnI5.35 ± 0.96 (1679) ** HindIII 1 (650) – HindIII HindIII 1.16 ± 0.08 (719) – The significance level for PstI indicates a significant decrease in transfection efficiency whereas all the other significance levels represent a significant increase. *, significant at 5% level; **, significant at 1% level, determined with the t-test. The enzymes used to digest the plasmid are in the left column and the enzymes added to the transformation mixture during transformation are in the next column. See Materials and Methods for details. There were no sites on the linear plasmid for enzymes added to the transformation mixture. The integration efficiency is represented as fold increase over the control experiments. The values are represented as the mean of 4–15 independent experiments ± standard deviation. Table 2. Effect of BamHI restrictionenzyme onREMIand colony-formingefficiencyofxrs5and CHO cells Cell line BamHI units Number of G418-resistant clones Colony-forming efficiency DNA+ buffer DNA+ BamHI DNA + buffer DNA + BamHI CHO 0 61 ± 0.7 64 ± 1.4 115 ± 7.1 124 ± 12.0 30 61 ± 6.4 116 ± 4.2 134 ± 5.7 127 ± 2.1 60 61 ± 2.1 128 ± 3.5 150 ± 2.1 109 ± 2.1 120 59 ± 0.7 110 ± 2.8 131 ± 12.0 118 ± 2.8 xrs5 033 ± 2.8 37 ± 9.1 54 ± 2.1 57 ± 4.9 30 35 ± 7.1 9 ± 2.1 50 ± 0.7 32 ± 4.9 60 33 ± 4.9 3 ± 1.4 50 ± 5.7 8 ± 2.8 120 32 + 1.4 1 ± 0.7 37 ± 2.8 2 ± 0.7 Plasmid PMA159 was digested with BamHI and linear DNA was electroporated into xrs5 and CHO cells. Integrants were selected in the medium containing G418. For the colony-forming efficiency, after electroporation, appropriate dilutions were made and plated without any selection. DNA with buffer is a control, without DNA no G418-resistant colony was obtained. The values are the mean of two experiments ± range. efficiency of illegitimate integration and also did not decrease plasmid pcDNA3.1/His/lacZ containing the lacZ gene was the efficiency of homologous integration. used. This plasmid was co-transfected into cells with or It was theoretically possible that the restriction enzymes, without the BglII enzyme and the frequency of blue cells was counted under the microscope. This frequency was 1.8% in the which increased the frequency of illegitimate integration, somehow inhibited uptake of the DNA into the cells. This presence of the enzyme and 1.3% in its absence. The difference would potentially cause a reduction in the frequency of was not significant. Thus, the restriction enzyme did not inhibit DNA uptake. homologous recombination, and the measured frequency of REMI events would be an underestimate. To address this ques- Since restriction enzymes show non-specific weak DNA tion, we performed a control experiment in which the linear binding(22), restrictionenzymes couldpossiblybindtothe 4830 Nucleic Acids Research, 2001, Vol. 29, No. 23 mouse cells, processing of the ends is in agreement with exonuclease activity degrading the ends before integration (23). In human cells, filling of PSS ends as well as loss of one to several hundred nucleotides was found in 24 out of 25 events during end joining (24). In fact, Derbyshire et al.(24) isolated such an end-joining activity tightly associated with the human homologous pairing activity and an intrinsic 3′–5′ exonuclease activity. The restriction enzymes BamHI, BglII and KpnI mediated integration events in yeast (2) as well as mammalian cells. HindIII was not active in yeast or mammalian cells in our experiment. In mouse Ltk– cells, however, 100 U of HindIII increased DNA integrations 4-fold (12), indicating that the 30 Uof HindIII we used might have been below the detection threshold for an effect of HindIII’s activity. EcoRI did not Figure 1. Substrate for homologous and illegitimate integration. A 7 kb increase the efficiency of DNA integration in yeast (2) whereas BamHI genomic fragment of Hprt containing exon 2 and 3 and introns 2 and 3 it was active in mammalian cells. The fact that three out of 10 was cloned into the BamHI site of PUC. The NEO gene was inserted into exon 3 (see Materials and Methods for details). Black boxes represent Hprt integration events, after transformation in the presence of sequences and gray boxes indicate neo and PUC sequences. A homologous EcoRI, in yeast were flanked by EcoRI sitessuggeststhat recombination event is shown on the left, upon which the phenotype of the EcoRI might possess low activity to mediate integrations in cells changes to G418 and 6-TG resistance. On the right, an IR event is shown, yeast, which is not sufficient to raise the integration efficiency (2). which renders the cells G418 resistant but they remain 6-TG sensitive. In Dictyostelium, BamHI, EcoRI, Sau3A, ClaIand BglII catalyzed integration of plasmids containing pyr5-6,a homolog of the yeast URA3 gene (25,26). In Cochliobolus heterostrophus, HindIII was usedtotag the TOX1 locus with transforming DNA, and in some way interfere with homology hygB (27). Thus, HindIII catalyzed an increase in integration search or otherwise inhibit homologous integration. To address events in Cochliobolus, but not in mammalian cells or yeast. this possibility we used purified BamHI-E77K protein, which These data indicate that the inability to raise the frequency of binds to the BamHI site but cleaves DNA at a rate 1000-fold integrations in our experiments may not be an intrinsic prop- lower than that of wild-type enzyme (22). The BamHI-E77K erty of some restriction enzymes, but may rather depend on protein did not increase the efficiency of illegitimate integra- cellular environment, and the ability to enter cells or different tion (Table 3) and did not appreciably decrease the frequency transformation conditions. The crystal structures of several of homologous gene targeting. Thus, only enzymes that enzymes have been identified and the active sites of BamHI, increase the efficiency of illegitimate integration also lead to a EcoRI and EcoRV are structurally similar (28,29). However, decrease in the frequency of gene targeting. their protein sequences are unrelated in most cases. Thus, within different cellular environments, some enzymes may be DISCUSSION better substrates for degradation through proteases than others. Some enzymes may not enter the mammalian cells, or even if Efficiency of REMI events with different enzymes they enter the cells, they may not be active in the nucleus Co-transfection of the restriction enzymes BamHI, BglII, because their ionic requirements for activity are not met. EcoRI and KpnI increased the efficiency of linearized plasmid In yeast, although digestion of a plasmid with Asp718 and integration up to 5-fold in CHO cells irrespective of the nature addition of BglII restriction enzyme to the transformation of the integrating DNA fragment ends (5′ or 3′ PSS or blunt mixtures significantly increased the efficiency of integration, ends). PstI, SmaIand HindIII restriction enzymes did not the integrating DNA was not inserted into the restriction sites induce REMI. In addition, the enzymes BamHI, BglII and (2). This suggested that the DNA breaks caused by the EcoRI increased integration efficiencies of transforming DNA enzymes open up the chromatin locally, which may lead to a in mouse ES cells. These results suggest that the restriction higher accessibility of the genomic DNA for integration. This enzymes are able to enter the nucleus and produce DNA may also be true for mammalian cells, which would also lead breaks. The repair process may lead to insertion of the DNA to the lack of recreation of the restriction sites flanking the substrate into these break sites. integrated vector. REMI has been characterized previously in yeast (1,2) and Electroporation of blunt end-producing endonucleases, there are several differences between the current data and the PvuII, EcoRV and StuI, caused toxicity and induced small previously published yeast data. First, in yeast, in the majority deletions of 1–36 bp, insertions, and combinations of inser- of REMI events the restriction sites at both sides of the inte- tions and deletions at the cleavage sites (30). Overexpression grated plasmid are recreated, indicating insertion of the of EcoRI enzyme in CHO cells revealed that 80–90% of the plasmid into genomic restriction sites without modification of surviving cells had chromosomal aberrations if the restriction the ends by simple annealing and ligation. In mammalian cells, enzyme is overexpressed in vivo for45min (13).Eventhough however, we find that the restriction sites are absent in the we did not see any toxic effect, we do not rule out the possi- majority of integration events. This is likely to be due to modi- bility of chromosomal aberrations in the transfected cells. fication of the ends before or during end joining. This is in Brenneman et al. (15) report that the plating efficiency of agreement with the previously published data (23,24). In human cells decreased by 80–90% after treatment of cells with Nucleic Acids Research, 2001, Vol. 29, No. 23 4831 Table 3. Effect of restriction enzymes on illegitimate and homologous integration of transfected DNA in ES cells Plasmid Enzyme added G418-resistant clones Fold increase 6-TG-resistant clones Homologous integration (%) +enzyme/–enzyme fraction J3N – 221 ± 38 1 9.8 ± 5.1 4.43 ± 2.30 – BamHI 893 ± 324 4.0 ± 1.4** 4.4 ± 1.3 0.49 ± 0.15 0.11 ± 0.05** HindIII 254 ± 31 1.1 ± 0.13 9.7 ± 0.6 3.8 ± 0.23 0.86 ± 0.056 BamHI-E77K 269 ± 21.1 1.2 ± 0.1 10.5 ± 2.2 3.9 ± 0.82 0.89 ± 0.019 J3NB – 253 ± 71 1 8.9 ± 4.7 3.44 ± 1.32 – BglII 925 ± 404 3.8 ± 1.8** 3.1 ± 1.6 0.37 ± 0.18 0.11 ± 0.06** J3NR – 197 ± 98 1 7.5 ± 4.4 3.72 ± 1.01 – EcoRI 347 ± 178 2.1 ± 0.75** 4.9 ± 2.7 1.51 ± 0.40 0.44 ± 0.19* This is the effect of the enzyme on the frequency of homologous integration events. All the plasmids were digested with BamHI and precipitated with ethanol. The linearized plasmid was electroporated into ES cells with enzyme or with shipping buffer alone in the controls (see details in Materials and Methods). Stable G418-resistant colonies expressing the integrated neo -containing plasmid result in 6-TG clones by integrating into the genomic HPRT locus. The values represent the mean and standard deviation of eight different cultures, except for HindIII (three different cultures) and BamHI-E77K (two cultures) where the mean ± range is given. **, significant at 1% level; *, significant at the 5% level. Significance was determined using the t-test for all enzymes except for BamHI-E77K. higher doses of restriction enzymes. Thus, it is likely that we Effect of restriction enzymes on gene targeting would also have seen toxicity at higher doses. We determined the effect of the BamHI enzyme on the frequency of homologous integration of an Hprt fragment Ku80 is required for REMI in mammalian cells flanked by BamHI sites. In the presence of restriction enzymes Addition of restriction enzymes during transformation with BamHI, BglII and EcoRI the fraction of gene-targeting events xrs5 cells showed no increase in the efficiency of DNA inte- among the overall number of integrants decreased up to 10-fold gration but the colony-forming efficiency was reduced consid- in ES cells. It is, however, well documented that DSBs can erably. At the highest concentration, most of the cells were stimulate homologous recombination in yeast and mammalian killed (Table 3). This suggests that Ku proteins are required for cells. DSBs and gaps in the region of homology carried by the integration of a vector into DSBs produced by restriction targeting plasmid stimulate recombination 33–140-fold in the enzymes. xrs5 cells are deficient in Ku80 function. In mamma- Hprt locus of ES cells (35). Homologous recombination rates lian cells, Ku proteins are needed for DSB repair and V(D)J at a given locus between homologs in somatic mammalian –8 –5 cells are generally low: 10 –10 per cell generation (36). recombination (6,7). Extrachromosomal homologous recombin- Frequencies of gene targeting are also low (37). Homologous ation and gene targeting by homologous recombination are not intrachromosomal recombination between partially duplicated affected in cells lacking Ku80 (31,32); however, end joining is Hprt genes of the human fibrosarcoma line HT1080 increased affected in those cells (33). Thus, Ku protein appears to play a by treatment with XbaIorI–SceI restriction endonucleases critical role only in the illegitimate end-joining pathway but (15). The recombination frequency increased only if the not in homologous recombination. Ku-deficient cells show restriction sites are present within the repeats. Furthermore, more DNA degradation at free ends of plasmids (33). This expression of I–SceI enhanced homologous recombination might account for a reduced frequency of REMI in our several thousand-fold between repeats at integrated defective experiments. The degradation of ends should, however, affect copies of the neomycin gene when the I–SceI site was present both spontaneous illegitimate integration and REMI to the in one of the repeats (32). Since BglII and EcoRI have sites in same extent. Since we found that REMI was completely the target, but those sites have been removed from the targeting abolished, and spontaneous illegitimate integration only vector, we expected that the frequency of homologous integra- decreased to ∼60% (Table 2), Ku is specifically implicated in tion would be enhanced. On the contrary, we found that the DSB-induced illegitimate integration. For spontaneous inte- frequency of homologous integration was 2-fold reduced after gration events, an alternative pathway, however, may exist in co-transformation of restriction enzymes. agreement with the data of Liang et al. (32). Other studies The result that the frequency of homologous recombination report a defect in plasmid integration in the xrs6 mutant, which was decreased 2-fold after co-transformation with restriction is dependent on the DNA concentration. At low concentration, enzymes is only possible if 100% of the cells that received the very little or no difference was observed between mutant and transforming plasmid also received the restriction enzyme. If wild-type cells, but at high plasmid concentration a 5–10-fold some cells receive the plasmid alone, without the restriction difference was found (34). In that study, the polybrene and enzyme, this decrease is an underestimate of the true effect. calcium phosphate methods were used, which are quite The degree of this underestimate depends on the number of different from the electroporation method that we used, which cells co-transfected. For instance, if 50% of the cells received makes a comparison of the DNA concentrations difficult. the plasmid alone the frequency of gene targeting needs to be 4832 Nucleic Acids Research, 2001, Vol. 29, No. 23 completely abolished in the remaining 50% of the cells that REFERENCES were co-transfected with both the restriction enzyme and the 1. Schiestl,R.H. and Petes,T.D. (1991) Integration of DNA fragments by plasmid to result in an ∼2-fold overall decrease. illegitimate recombination in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA, 88, 7585–7589. Most previous experiments suggest that the illegitimate end- 2. Manivasakam,P. and Schiestl,R.H. 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We have shown that restriction enzymes can increase the USA, 93, 3608–3612. frequency of illegitimate integration in mammalian cells, 16. Sargent,R.G., Brenneman,M.A. and Wilson,J.H. (1997) Repair of which is dependent on Ku80. Furthermore, restriction enzymes site-specific double-strand breaks in a mammalian chromosome by that increased the frequency of illegitimate integration, at the homologous and illegitimate recombination. Mol. Cell. Biol., 17, 267–277. 17. Zhang,S.Z. and Dong,W.F. (1987) Chromosomal aberrations induced by same time, reduced the frequency of gene targeting, suggesting the restriction endonucleases Eco RI, Pst I, Sal I and Bam HI in CHO that the two pathways are directly competing for the recombi- cells. Mutat. Res., 180, 109–114. nation substrate. Characterizing the genetically, endogenously 18. Vasudev,V. and Obe,G. (1987) Effect of heat treatment on chromosomal and possibly exogenously introduced factors modifying both aberrations induced by the alkylating agent trenimon or the restriction recombination pathways in this competition may lead to better endonuclease Alu I in Chinese hamster ovary (CHO) cells. Mutat. Res., 178, 81–90. understanding of this process and possibly to the advancements 19. Natarajan,A.T. and Obe,G. (1984) Molecular mechanisms involved in the of gene targeting. production of chromosomal aberrations. III. Restriction endonucleases. Chromosoma, 90, 120–127. 20. Schiestl,R.H., Dominska,M. and Petes,T.D. (1993) Transformation of ACKNOWLEDGEMENTS Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo We thank the members of the Schiestl laboratory for helpful ligation of transforming DNA to mitochondrial DNA sequences. suggestions and discussion. We also thank Penny Jeggo (MRC Mol. Cell. Biol., 13, 2697–2705. Cell Mutation Unit, University of Sussex, UK) for the 21. Bryant,P.E. (1988) Use of restriction endonucleases to study relationships generous gift of the xrs5 CHO mutant cells and Nobuyo Meada between DNA double-strand breaks, chromosomal aberrations and other end-points in mammalian cells. Int. J. Radiat. Biol., 54, 869–890. (University of North Carolina, Chapel Hill, NC) for the mouse 22. Xu,S.Y. and Schildkraut,I. (1991) Cofactor requirements of BamHI Hprt fragment. Supported by grant No. CN-83B from the mutant endonuclease E77K and its suppressor mutants. J. Bacteriol., 173, American Cancer Society and Research Career Development 5030–5035. Award ES00299 from the National Institute of Environmental 23. Henderson,G. and Simons,J.P. (1997) Processing of DNA prior to Health Sciences to R.H.S. illegitimate recombination in mouse cells. Mol. Cell. Biol., 17, 3779–3785. Nucleic Acids Research, 2001, Vol. 29, No. 23 4833 24. Derbyshire,M.K., Epstein,L.H., Young,C.S., Munz,P.L. and Fishel,R. 33. Liang,F. and Jasin,M. (1996) Ku80-deficient cells exhibit excess (1994) Nonhomologous recombination in human cells. Mol. Cell. Biol., degradation of extrachromosomal DNA. J. Biol. Chem., 271, 14405–14411. 14, 156–169. 34. Jeggo,P.A. and Smith-Ravin,J. (1989) Decreased stable transfection 25. Kuspa,A. and Loomis,W.F. (1992) Tagging developmental genes in frequencies of six X-ray-sensitive CHO strains, all members of the xrs Dictyostelium by restriction enzyme-mediated integration of plasmid complementation group. Mutat. Res., 218, 75–86. DNA. Proc. Natl Acad. Sci. USA, 89, 8803–8807. 35. Valancius,V. and Smithies,O. (1991) Double-strand gap repair in a 26. Kuspa,A. and Loomis,W.F. (1994) REMI-RFLP mapping in the mammalian gene-targeting reaction. Mol. Cell. Biol., 11, 4389–4397. Dictyostelium genome. Genetics, 138, 665–674. 36. Bollag,R.J., Waldman,A.S. and Liskay,R.M. (1989) Homologous 27. Lu,S., Lyngholm,L., Yang,G., Bronson,C., Yoder,O.C. and Turgeon,B.G. recombination in mammalian cells. Annu.Rev.Genet., 23, 199–225. (1994) Tagged mutations at the Tox1 locus of Cochliobolus 37. Hasty,P., Rivera-Perez,J., Chang,C. and Bradley,A. (1991) Target heterostrophus by restriction enzyme-mediated integration. frequency and integration pattern for insertion and replacement vectors in Proc. Natl Acad. Sci. USA, 91, 12649–12653. embryonic stem cells. Mol. Cell. Biol., 11, 4509–4517. 28. Winkler,F.K., Banner,D.W., Oefner,C., Tsernoglou,D., Brown,R.S., 38. Lukacsovich,T., Yang,D. and Waldman,A.S. (1994) Repair of a specific Heathman,S.P., Bryan,R.K., Martin,P.D., Petratos,K. and Wilson,K.S. double-strand break generated within a mammalian chromosome by yeast (1993) The crystal structure of EcoRV endonuclease and of its complexes endonuclease I-SceI. Nucleic Acids Res., 22, 5649–5657. with cognate and non-cognate DNA fragments. EMBO J., 12, 1781–1795. 39. Carroll,D. (1983) Genetic recombination of bacteriophage lambda DNAs 29. Newman,M., Strzelecka,T., Dorner,L.F., Schildkraut,I. and in Xenopus oocytes. Proc. Natl Acad. Sci. USA, 80, 6902–6906. Aggarwal,A.K. (1994) Structure of restriction endonuclease BamHI and 40. Carroll,D., Wright,S.H., Wolff,R.K., Grzesiuk,E. and Maryon,E.B. (1986) its relationship to EcoRI. Nature, 368, 660–664. Efficient homologous recombination of linear DNA substrates after 30. Phillips,J.W. and Morgan,W.F. (1994) Illegitimate recombination induced injection into Xenopus laevis oocytes. Mol. Cell. Biol., 6, 2053–2061. by DNA double-strand breaks in a mammalian chromosome. Mol. Cell. Biol., 41. Goedecke,W., Vielmetter,W. and Pfeiffer,P. (1992) Activation of a 14, 5794–5803. system for the joining of nonhomologous DNA ends during Xenopus egg 31. Morrison,C. and Wagner,E. (1996) Extrachromosomal recombination maturation. Mol. Cell. Biol., 12, 811–816. occurs efficiently in cells defective in various DNA repair systems. 42. Pfeiffer,P. and Vielmetter,W. (1988) Joining of nonhomologous DNA Nucleic Acids Res., 24, 2053–2058. double strand breaks in vitro. Nucleic Acids Res., 16, 907–924. 32. Liang,F., Romanienko,P.J., Weaver,D.T., Jeggo,P.A. and Jasin,M. (1996) Chromosomal double-strand break repair in Ku80-deficient cells. 43. Rusconi,S. and Schaffner,W. (1981) Transformation of frog embryos with Proc. Natl Acad. Sci. USA, 93, 8929–8933. a rabbit β-globin gene. Proc. Natl Acad. Sci. USA, 78, 5051–5055. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells

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0305-1048
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10.1093/nar/29.23.4826
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Abstract

4826–4833 Nucleic Acids Research, 2001, Vol. 29, No. 23 © 2001 Oxford University Press Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells Palaniyandi Manivasakam, Jiri Aubrecht, Samy Sidhom and Robert H. Schiestl* Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA Received August 8, 2001; Revised and Accepted October 18, 2001 ABSTRACT which may be lethal. Inappropriate repair of DSBs can result in chromosomal rearrangements, which can lead to cell Mammalian cells repair DNA double-strand breaks by transformation. illegitimate end-joining or by homologous recombin- Saccharomyces cerevisiae cells repair DSBs by homologous ation. We investigated the effects of restriction recombination, mainly mediated by gene products in the enzymes on illegitimate and homologous DNA RAD52 pathway. In the absence of RAD52, yeast cells repair integration in mammalian cells. A plasmid containing DSBs by non-homologous end joining (NHEJ), which requires the neo expression cassette, which confers G418 the Ku homologous proteins. NHEJ has been studied in yeast amongst other approaches by transformation with a linear resistance, was used to select for illegitimate integra- DNA molecule that contains a selectable marker, which does tion events in CHO wild-type and xrcc5 mutant cells. not have any homology to the genome. Schiestl and Petes (1) Co-transfection with the restriction enzymes BamHI, used a BamHI fragment containing the URA3 marker to trans- BglII, EcoRI and KpnI increased the efficiency of form a yeast strain in which the entire URA3 gene had been linearized plasmid integration up to 5-fold in CHO deleted. After addition of the BamHI restriction enzyme to the cells. In contrast, the restriction enzymes did not transformation mixture the efficiency of integration increased increase the integration efficiency in xrcc5 mutant several-fold and integrations occurred into BamHI sites. These cells. Effects of restriction enzymes on illegitimate events were designated restriction enzyme-mediated integra- and homologous integration were also studied in tion (REMI) events. This study was extended to investigate mouse embryonic stem (ES) cells using a plasmid how two different compatible and non-compatible ends are containing the neo gene flanked by exon 3 of Hprt. repaired (2), by restricting the DNA with one enzyme and adding another enzyme to the transformation mixture. The enzymes BamHI, BglII and EcoRI increased the Mammalian cells repair DSBs both by homologous recombin- illegitimate integration efficiency of transforming ation and by illegitimate recombination (IR). However, DNA several-fold, similar to the results for CHO cells. homologous integration frequencies are 100–1000-fold less However, all three enzymes decreased the absolute frequent than illegitimate integration (3,4), the major obstacle frequency of homologous integration ∼2-fold, and for gene targeting in mammalian cells. The molecular mechanism the percentage of homologous integration decreased of recombination in mammalian cells is under intensive >10-fold. This suggests that random DNA breaks investigation. Different complementation groups of ionizing attract illegitimate recombination (IR) events that radiation-sensitive rodent cell mutants have been identified and compete with homology search. three of them designated X-ray repair cross-complementing (XRCC) groups 5, 6 and 7 (5). The genes defective in groups 5 and 7, deficient in the mutants xrcc5 and xrcc7, respectively, INTRODUCTION encode components of a DNA-dependent protein kinase In the eukaryotic genome, DNA double-strand breaks (DSBs) (DNA-PK), a complex possessing DNA end-binding and can occur during cellular processes such as DNA repair, protein kinase activity (6–8). The DNA-binding subunit of recombination and replication; the early prophase of meiosis, DNA-PK is a heterodimer of 70- and 80-kDa subunits, named V(D)J recombination or as the result of exposure to DNA Ku70 and Ku80, respectively. Ku80 is deficient in group damaging agents. The repair of DSBs is important for the 5 cells (9). Ku is an abundant nuclear protein identified originally maintenance of genomic integrity and cellular survival because as an autoantigen from various autoimmune patients (10). The unrepaired DSBs will result in the loss of genetic information, Ku protein binds to free double-stranded DNA ends with 5′-or *To whom correspondence should be addressed at present address: Departments of Pathology and Environmental Health, UCLA School of Medicine and Public Health, 650 Charles E. Young Drive South, 71-295 CHS, Los Angeles, CA 90095, USA. Tel: +1 310 267 2087; Fax: +1 310 267 2578; Email: rschiestl@mednet.ucla.edu Present addresses: Palaniyandi Manivasakam, CombinatoRx, 650 Albany Street, Boston, MA 02118, USA Jiri Aubrecht, Pfizer Global Research and Development, Groton, CT 06340, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors Nucleic Acids Research, 2001, Vol. 29, No. 23 4827 3′-single-stranded protruding (PSS) ends or blunt ends, nicked from the 5′ end) in plasmid J3N. Plasmid pcDNA3.1/His/lacZ DNA, and duplex DNA ending with stem–loop structures (10), (Invitrogen, Carlsbad, CA) control vector has been used for which suggests a possible role in DNA repair and recombination. transient transfection to determine the effect of restriction These mutants are defective in DSB rejoining and have been enzymes on DNA uptake. shown to possess defects in components of DNA-PK. They Cell lines also exhibit defects in the rejoining steps in V(D)J recombination, a site-specific recombination process required for rearrangement CHO cells (CHO-K1; ATCC) and the xrs5 mutant cells of DNA to generate variability in genes encoding immuno- (obtained from Penny Jeggo, MRC Cell Mutation Unit, globulins. University of Sussex, UK) were grown in Cellgro DMEM Several investigators have utilized restriction enzymes to (Mediatech, Herndon, VA) supplemented with proline (3.7 mM), study the effect of DSB repair in mammalian cells. It has been 10% fetal bovine serum (Life Technologies, Rockville, MD), shown that introduction of restriction enzymes into mamma- 100 U/ml penicillin and 100 µ g/ml streptomycin in a humidified lian cells, either by electroporation or by calcium precipitation, air incubator at 37°C, 5% CO and 95% humidity. Embryonic produces cell killing, chromosomal aberrations, gene amplifi- stem (ES) cells (ES-D3; ATTC) were cultivated on monolayers of cation and increased mutation frequency at several loci [for irradiated feeder cells (STO-TN) in DMEM supplemented with review (11)]. Co-transfection with restriction enzymes HindIII 10% fetal bovine serum (Life Technologies), LIF (100 U/ml), and XbaI induces DNA integration into mouse Ltk cells (12). 0.2 mM mercaptoethanol (Life Technologies), 100 U/ml penicillin Inducible expression of EcoRI in CHO cells showed toxicity and 100 µ g/ml streptomycin (Mediatech) in a humidified air and chromosomal aberrations (13). incubator at 37°C, 5% CO and 95% humidity. The feeder cell line was developed from embryonic fibroblast cells (STO; Investigators showed that intrachromosomal homologous ATTC). That feeder cell line was transfected with plasmid PMA159 recombination can be induced by electroporation with restric- and selected in the presence of G418 (Life Technologies). One tion enzymes or intracellular expression of restriction enzymes stable transfected clone of STO fibroblast cells (STO-TN) (14,15). Using a loss of function assay in the APRT gene, conferring resistance to 1 mg/ml G418 was selected for prepara- Sargent et al. (16) showed that DSBs created by I–SceI tion of feeder cells. The STO-TN cell line carries a mutated induced homologous recombination 100-fold between repeats, hprt gene and neo expression cassette that allows simulta- but IR was stimulated 1000-fold. This indicates that the prefer- neous selection with 6-thioguanine (6-TG) and G418. A 90% ential mode of DSB repair in mammalian cells is NHEJ. confluent culture of STO cells was trypsinized, suspended in In the present study, we investigated the effects of restriction DMEM and irradiated with a 50 Gy dose of γ irradiation with a enzymes on illegitimate integration and homologous gene Co γ-ray source at a dose rate of 12.2 cGY/s. After irradiation, targeting in mammalian cells. We found that restriction the cells were frozen until further use. The frozen feeder cells were enzymes increased the frequency of illegitimate integration, thawed 3–10 days before seeding of ES cells, then plated at a which was dependent on the Ku80 function. Surprisingly, we density of 2.5–4 × 10 cells per 10 cm dish and grown to near also found that the restriction enzymes decreased the confluency. frequency of homologous integration, indicating a direct competition between the two pathways. Electroporation of cells CHO or xrs5 cells were grown to 90% confluence in 225 cm MATERIALS AND METHODS culture flasks (Corning, Corning, NY) and trypsinized using trypsin/EDTA (Mediatech). The detached cells were collected Plasmids in 10 ml of fresh culture medium, washed twice with phosphate- Plasmid PMA159 was derived from pUC and contains the neo buffered saline (PBS) and resuspended in electroporation gene. This plasmid was constructed in two steps. Digestion of buffer (272 mM sucrose, 7 mM Na HPO ,1mM MgCl ,pH 7.4). 2 4 2 pBK-CMV plasmid (from Stratagene) with AvrII restriction Approximately 3–5 × 10 cells in 0.8 ml of electroporation enzyme liberated the 1.6 kb neo expression cassette. BamHI buffer were mixed with 15 µ g of linearized plasmid linkers were ligated to the ends of the fragment after filling in (PMA159), 30–480 U of restriction enzyme or an appropriate the 5′ single-stranded ends with Klenow. neo was inserted amount of restriction enzyme buffer and restriction enzyme into the BamHI site of pUC. After partial digestion, the 5′ end storage buffer. The electroporation was performed using Bio-Rad of the BamHI site was eliminated by filling in the single-stranded Gene Pulser at 0.3 kV, 960 µ Fand 200 Ω . The electroporated ends and ligating the blunt ends. Plasmid J3N contains the neo cells were diluted with fresh culture medium, plated on to three gene flanked by exon 3 of the Hprt gene. A 7 kb BamHI frag- 10 cm dishes (Falcon) and incubated. After 24 h, the medium ment containing exons 2 and 3 of the Hprt gene was ligated was supplemented with G418 to a final concentration of 750 µ M. into pUC19 missing an EcoRI site. The neo expression To determine the viability after electroporation, 100 and 500 cells cassette obtained by AvrII digestion was blunt ended with were plated on to two 10 cm dishes. To minimize the occurrence Klenow, and XhoI linker was attached. The neo fragment of satellite clones the cultures were incubated without disturbance containing XhoI ends was ligated into the XhoI site, which for 11–14 days. The colonies of surviving cells were stained residesinexon3ofthe Hprt gene, yielding plasmid J3N. This using Giemsa stain (Sigma, St Louis, MO). plasmidhas homology to Hprt of 4.5and 2.5kbflankingthe ES cells were growntoconfluenceon10cmdishes covered neo fragment. J3NB plasmid was constructed by filling in two with STO-TN feeder cells. After trypsinization the cells BglII sites (located 1 and 3.5 kb from the 5′ end), J3NR by were resuspended in fresh medium and washed twice in filling in the two EcoRI sites(located4and 5.5kbfrom the PBS. Approximately 8–12 × 10 cells were electroporated in 5′ end) andJ3NH byfilling inthe HindIII site (located 3.7 kb PBS using Bio-Rad Gene Pulser at 0.3 kV and 500 µ F. The 4828 Nucleic Acids Research, 2001, Vol. 29, No. 23 electroporated ES cells were seeded on three 10 cm dishes producing PSS ends (19), was used. Except for PvuII, which covered with STO-TN feeder cells. After 24 h of incubation, showed a 50% reduction in colony forming unit (CFU), none the medium was supplemented with 500 µ g/ml G418. The of the other enzymes showed any significant level of reduction surviving colonies of stable integrants including random and in CFU with 30 U (data not shown). At the highest concentra- homologous recombination events were counted after 10 days. tion of 480 U, the enzymes BglII and PvuII, but none of the At that time, the medium was replaced with one containing other enzymes used including PstI, decreased the CFU 30 µ M 6-thioguanine (TG) (Sigma). Surviving colonies were significantly (data not shown). These results indicate that the counted after 5–7 days. restriction enzymes that were used for the REMI experiment were not toxic to the mammalian cells at the concentration Transient transfection to determine DNA uptake used for integration studies (30 U). Plasmid pcDNA3.1/His/lacZ (Invitrogen) was digested with REMI events in yeast usually integrate into restriction sites BglII, and cells were prepared and electroporated with the in the genome by micro-homology-mediated integration, and linear plasmid as described above in the presence or absence of both restriction sites at the ends of the integrating DNA are the BglII enzyme. The control in the absence of the enzyme maintained (1,20). This facilitates detection of such an event contained an equivalent amount of the enzyme storage buffer. simply by digestion of the genomic DNA with the same restric- The cells were then incubated overnight in the presence of tion enzyme that was used for REMI, and performing a X-gal. Cells were counted and screened for blue color under Southern blot that reveals the fragment size of the integrating the microscope. vector. We performed Southern blot analysis with twelve clones obtained after a transformation with the BamHI- Southern blot analysis digested plasmid PMA159 in the presence of BamHI (data not Stable transformants were obtained by transforming the cells shown). This experiment indicates that a single copy integra- with PMA159 plasmid, which was restricted with BamHI and tion event recreated both flanking BamHI sites inonly1 out of transfected with or without BamHI enzyme or with the plasmid 12 transformed clones. In the control in which no enzyme was digested with KpnI and transfected with or without KpnI. added, none of the events was flanked by BamHI sites. In the Individual clones were expanded by growing in 10 cm dishes. experiment with KpnI, 1 out of 10 events recreated the flanking The genomic DNA was isolated and Southern blot analyses KpnI sites, and in the control, none of the events recreated both were performed according to standard procedures using the KpnI sites. Control cells as well as restriction enzyme-treated 1.6 kb neo gene as probe. cells showed multiple fragments of various sizes, suggesting multiple copies at a single site and/or multiple integration events at multiple sites. RESULTS It has been reported that radiosensitive cells are more susceptible to the toxic effects and chromosome aberrations We investigated the effects of restriction enzymes on illegiti- caused by restriction enzymes (21). We used the xrs5 cell line mate and homologous DNA integrations in mammalian cells. that is deficient in Ku80 to study the toxicity and integration- To study IR, a plasmid containing the neo selection marker mediating effect of restriction enzymes. Approximately 55% was used. A DNA fragment containing the neo gene was of the cells survived with 30 U of the BamHI enzyme, while cloned into the BamHI site of pUC plasmid and one BamHI only 14% survived with 60 U (Table 2). In the presence of 30 site was eliminated by filling in and religation. All multi- and60U of the BamHI enzyme, REMI efficiency decreased to cloning sites are unique in this plasmid. The plasmid was ∼25 and 8%, respectively, in xrs5 cells, which is a greater linearized with various restriction enzymes and transfected in decrease than caused by toxicity alone (Table 2). Thus, Ku80 the presence of enzyme into the cells by electroporation. seems to be required for REMI. Initially we determined the effects of different concentrations (30, 120 and 480 U per transfection vial) of the BamHI enzyme Next, we investigated the effects of restriction enzymes on on the frequency of integration. The same increase in the the efficiency of gene targeting in ES cells. For this study, we efficiency of integration was seen with all three concentrations, constructed a plasmid containing the neo gene as a selec- and thus, 30 U were used for all further experiments. Since tion marker flanked by the Hprt gene (Fig. 1). This plasmid restriction enzyme buffers can produce chromosomal aberrations can be used to select for illegitimate integration as well as (17,18), the same concentration of restriction enzyme buffer for gene targeting. Integration of the plasmid will give rise and storage buffer as used for the restriction digests was used to G418-resistant clones. Homologous integration results in in all controls. The restriction enzymes BamHI, BglII and disruption of the Hprt gene, which can be selected for with EcoRI, which produce 5′ PSS ends, and KpnI, which produces 6-TG. As the hprt locus is hemizygous in XY ES cells, 3′ PSS ends, increased the efficiency of illegitimate integration targeted clones can be isolated by direct selection in 6-TG 2.5–5-fold (Table 1) in CHO cells. PstI, SmaIand HindIII for lack of hprt function. We tested the effect of BamHI, restriction enzymes, producing 3′ PSS ends, blunt ends and BamHI-E77K, BglII, EcoRI and HindIII restriction enzymes 5′ PSS ends, respectively, did not induce REMI. on gene targeting in ES cells. In the presence of BamHI, BglII Addition of PstI decreased the integration efficiency 2-fold. and EcoRI the total number of integration events increased We wanted to determine whether this decrease was due to 2–4-fold as seen in CHO cells (Table 3). Surprisingly, possible toxic effects of the enzyme. The cells were transfected however, in the presence of enzyme, the actual number of with three different concentrations: 30, 120 and 480 U for each homologous integration events decreased >2-fold, and the of the enzymes used in this study. As a control, PvuII, which is percentage of homologous integration as a fraction of all known to be a more potent inducer of toxic effects and integration events decreased down to >10-fold (Table 3). The chromosomal aberrations in mammalian cells than enzymes addition of HindIII, on the other hand, did not increase the Nucleic Acids Research, 2001, Vol. 29, No. 23 4829 Table 1. Effect of different restriction enzymes on REMI in CHO cells Plasmid digested with Enzyme added Integration (fold over control) No. of clones Significance BamHI – 1 (298) BamHI BamHI 3.74 ± 0.28 (1009) ** BglII – 1 (177) BglII BglII 5.49 ± 0.49 (928) ** EcoRI – 1 (223) – EcoRI EcoRI 2.40 ± 0.24 (479) ** PstI – 1 (935) – PstI PstI0.46 ± 0.05 (420) ** SmaI – 1 (254) – SmaI SmaI0.84 ± 0.05 (233) – KpnI – 1 (445) – KpnI KpnI5.35 ± 0.96 (1679) ** HindIII 1 (650) – HindIII HindIII 1.16 ± 0.08 (719) – The significance level for PstI indicates a significant decrease in transfection efficiency whereas all the other significance levels represent a significant increase. *, significant at 5% level; **, significant at 1% level, determined with the t-test. The enzymes used to digest the plasmid are in the left column and the enzymes added to the transformation mixture during transformation are in the next column. See Materials and Methods for details. There were no sites on the linear plasmid for enzymes added to the transformation mixture. The integration efficiency is represented as fold increase over the control experiments. The values are represented as the mean of 4–15 independent experiments ± standard deviation. Table 2. Effect of BamHI restrictionenzyme onREMIand colony-formingefficiencyofxrs5and CHO cells Cell line BamHI units Number of G418-resistant clones Colony-forming efficiency DNA+ buffer DNA+ BamHI DNA + buffer DNA + BamHI CHO 0 61 ± 0.7 64 ± 1.4 115 ± 7.1 124 ± 12.0 30 61 ± 6.4 116 ± 4.2 134 ± 5.7 127 ± 2.1 60 61 ± 2.1 128 ± 3.5 150 ± 2.1 109 ± 2.1 120 59 ± 0.7 110 ± 2.8 131 ± 12.0 118 ± 2.8 xrs5 033 ± 2.8 37 ± 9.1 54 ± 2.1 57 ± 4.9 30 35 ± 7.1 9 ± 2.1 50 ± 0.7 32 ± 4.9 60 33 ± 4.9 3 ± 1.4 50 ± 5.7 8 ± 2.8 120 32 + 1.4 1 ± 0.7 37 ± 2.8 2 ± 0.7 Plasmid PMA159 was digested with BamHI and linear DNA was electroporated into xrs5 and CHO cells. Integrants were selected in the medium containing G418. For the colony-forming efficiency, after electroporation, appropriate dilutions were made and plated without any selection. DNA with buffer is a control, without DNA no G418-resistant colony was obtained. The values are the mean of two experiments ± range. efficiency of illegitimate integration and also did not decrease plasmid pcDNA3.1/His/lacZ containing the lacZ gene was the efficiency of homologous integration. used. This plasmid was co-transfected into cells with or It was theoretically possible that the restriction enzymes, without the BglII enzyme and the frequency of blue cells was counted under the microscope. This frequency was 1.8% in the which increased the frequency of illegitimate integration, somehow inhibited uptake of the DNA into the cells. This presence of the enzyme and 1.3% in its absence. The difference would potentially cause a reduction in the frequency of was not significant. Thus, the restriction enzyme did not inhibit DNA uptake. homologous recombination, and the measured frequency of REMI events would be an underestimate. To address this ques- Since restriction enzymes show non-specific weak DNA tion, we performed a control experiment in which the linear binding(22), restrictionenzymes couldpossiblybindtothe 4830 Nucleic Acids Research, 2001, Vol. 29, No. 23 mouse cells, processing of the ends is in agreement with exonuclease activity degrading the ends before integration (23). In human cells, filling of PSS ends as well as loss of one to several hundred nucleotides was found in 24 out of 25 events during end joining (24). In fact, Derbyshire et al.(24) isolated such an end-joining activity tightly associated with the human homologous pairing activity and an intrinsic 3′–5′ exonuclease activity. The restriction enzymes BamHI, BglII and KpnI mediated integration events in yeast (2) as well as mammalian cells. HindIII was not active in yeast or mammalian cells in our experiment. In mouse Ltk– cells, however, 100 U of HindIII increased DNA integrations 4-fold (12), indicating that the 30 Uof HindIII we used might have been below the detection threshold for an effect of HindIII’s activity. EcoRI did not Figure 1. Substrate for homologous and illegitimate integration. A 7 kb increase the efficiency of DNA integration in yeast (2) whereas BamHI genomic fragment of Hprt containing exon 2 and 3 and introns 2 and 3 it was active in mammalian cells. The fact that three out of 10 was cloned into the BamHI site of PUC. The NEO gene was inserted into exon 3 (see Materials and Methods for details). Black boxes represent Hprt integration events, after transformation in the presence of sequences and gray boxes indicate neo and PUC sequences. A homologous EcoRI, in yeast were flanked by EcoRI sitessuggeststhat recombination event is shown on the left, upon which the phenotype of the EcoRI might possess low activity to mediate integrations in cells changes to G418 and 6-TG resistance. On the right, an IR event is shown, yeast, which is not sufficient to raise the integration efficiency (2). which renders the cells G418 resistant but they remain 6-TG sensitive. In Dictyostelium, BamHI, EcoRI, Sau3A, ClaIand BglII catalyzed integration of plasmids containing pyr5-6,a homolog of the yeast URA3 gene (25,26). In Cochliobolus heterostrophus, HindIII was usedtotag the TOX1 locus with transforming DNA, and in some way interfere with homology hygB (27). Thus, HindIII catalyzed an increase in integration search or otherwise inhibit homologous integration. To address events in Cochliobolus, but not in mammalian cells or yeast. this possibility we used purified BamHI-E77K protein, which These data indicate that the inability to raise the frequency of binds to the BamHI site but cleaves DNA at a rate 1000-fold integrations in our experiments may not be an intrinsic prop- lower than that of wild-type enzyme (22). The BamHI-E77K erty of some restriction enzymes, but may rather depend on protein did not increase the efficiency of illegitimate integra- cellular environment, and the ability to enter cells or different tion (Table 3) and did not appreciably decrease the frequency transformation conditions. The crystal structures of several of homologous gene targeting. Thus, only enzymes that enzymes have been identified and the active sites of BamHI, increase the efficiency of illegitimate integration also lead to a EcoRI and EcoRV are structurally similar (28,29). However, decrease in the frequency of gene targeting. their protein sequences are unrelated in most cases. Thus, within different cellular environments, some enzymes may be DISCUSSION better substrates for degradation through proteases than others. Some enzymes may not enter the mammalian cells, or even if Efficiency of REMI events with different enzymes they enter the cells, they may not be active in the nucleus Co-transfection of the restriction enzymes BamHI, BglII, because their ionic requirements for activity are not met. EcoRI and KpnI increased the efficiency of linearized plasmid In yeast, although digestion of a plasmid with Asp718 and integration up to 5-fold in CHO cells irrespective of the nature addition of BglII restriction enzyme to the transformation of the integrating DNA fragment ends (5′ or 3′ PSS or blunt mixtures significantly increased the efficiency of integration, ends). PstI, SmaIand HindIII restriction enzymes did not the integrating DNA was not inserted into the restriction sites induce REMI. In addition, the enzymes BamHI, BglII and (2). This suggested that the DNA breaks caused by the EcoRI increased integration efficiencies of transforming DNA enzymes open up the chromatin locally, which may lead to a in mouse ES cells. These results suggest that the restriction higher accessibility of the genomic DNA for integration. This enzymes are able to enter the nucleus and produce DNA may also be true for mammalian cells, which would also lead breaks. The repair process may lead to insertion of the DNA to the lack of recreation of the restriction sites flanking the substrate into these break sites. integrated vector. REMI has been characterized previously in yeast (1,2) and Electroporation of blunt end-producing endonucleases, there are several differences between the current data and the PvuII, EcoRV and StuI, caused toxicity and induced small previously published yeast data. First, in yeast, in the majority deletions of 1–36 bp, insertions, and combinations of inser- of REMI events the restriction sites at both sides of the inte- tions and deletions at the cleavage sites (30). Overexpression grated plasmid are recreated, indicating insertion of the of EcoRI enzyme in CHO cells revealed that 80–90% of the plasmid into genomic restriction sites without modification of surviving cells had chromosomal aberrations if the restriction the ends by simple annealing and ligation. In mammalian cells, enzyme is overexpressed in vivo for45min (13).Eventhough however, we find that the restriction sites are absent in the we did not see any toxic effect, we do not rule out the possi- majority of integration events. This is likely to be due to modi- bility of chromosomal aberrations in the transfected cells. fication of the ends before or during end joining. This is in Brenneman et al. (15) report that the plating efficiency of agreement with the previously published data (23,24). In human cells decreased by 80–90% after treatment of cells with Nucleic Acids Research, 2001, Vol. 29, No. 23 4831 Table 3. Effect of restriction enzymes on illegitimate and homologous integration of transfected DNA in ES cells Plasmid Enzyme added G418-resistant clones Fold increase 6-TG-resistant clones Homologous integration (%) +enzyme/–enzyme fraction J3N – 221 ± 38 1 9.8 ± 5.1 4.43 ± 2.30 – BamHI 893 ± 324 4.0 ± 1.4** 4.4 ± 1.3 0.49 ± 0.15 0.11 ± 0.05** HindIII 254 ± 31 1.1 ± 0.13 9.7 ± 0.6 3.8 ± 0.23 0.86 ± 0.056 BamHI-E77K 269 ± 21.1 1.2 ± 0.1 10.5 ± 2.2 3.9 ± 0.82 0.89 ± 0.019 J3NB – 253 ± 71 1 8.9 ± 4.7 3.44 ± 1.32 – BglII 925 ± 404 3.8 ± 1.8** 3.1 ± 1.6 0.37 ± 0.18 0.11 ± 0.06** J3NR – 197 ± 98 1 7.5 ± 4.4 3.72 ± 1.01 – EcoRI 347 ± 178 2.1 ± 0.75** 4.9 ± 2.7 1.51 ± 0.40 0.44 ± 0.19* This is the effect of the enzyme on the frequency of homologous integration events. All the plasmids were digested with BamHI and precipitated with ethanol. The linearized plasmid was electroporated into ES cells with enzyme or with shipping buffer alone in the controls (see details in Materials and Methods). Stable G418-resistant colonies expressing the integrated neo -containing plasmid result in 6-TG clones by integrating into the genomic HPRT locus. The values represent the mean and standard deviation of eight different cultures, except for HindIII (three different cultures) and BamHI-E77K (two cultures) where the mean ± range is given. **, significant at 1% level; *, significant at the 5% level. Significance was determined using the t-test for all enzymes except for BamHI-E77K. higher doses of restriction enzymes. Thus, it is likely that we Effect of restriction enzymes on gene targeting would also have seen toxicity at higher doses. We determined the effect of the BamHI enzyme on the frequency of homologous integration of an Hprt fragment Ku80 is required for REMI in mammalian cells flanked by BamHI sites. In the presence of restriction enzymes Addition of restriction enzymes during transformation with BamHI, BglII and EcoRI the fraction of gene-targeting events xrs5 cells showed no increase in the efficiency of DNA inte- among the overall number of integrants decreased up to 10-fold gration but the colony-forming efficiency was reduced consid- in ES cells. It is, however, well documented that DSBs can erably. At the highest concentration, most of the cells were stimulate homologous recombination in yeast and mammalian killed (Table 3). This suggests that Ku proteins are required for cells. DSBs and gaps in the region of homology carried by the integration of a vector into DSBs produced by restriction targeting plasmid stimulate recombination 33–140-fold in the enzymes. xrs5 cells are deficient in Ku80 function. In mamma- Hprt locus of ES cells (35). Homologous recombination rates lian cells, Ku proteins are needed for DSB repair and V(D)J at a given locus between homologs in somatic mammalian –8 –5 cells are generally low: 10 –10 per cell generation (36). recombination (6,7). Extrachromosomal homologous recombin- Frequencies of gene targeting are also low (37). Homologous ation and gene targeting by homologous recombination are not intrachromosomal recombination between partially duplicated affected in cells lacking Ku80 (31,32); however, end joining is Hprt genes of the human fibrosarcoma line HT1080 increased affected in those cells (33). Thus, Ku protein appears to play a by treatment with XbaIorI–SceI restriction endonucleases critical role only in the illegitimate end-joining pathway but (15). The recombination frequency increased only if the not in homologous recombination. Ku-deficient cells show restriction sites are present within the repeats. Furthermore, more DNA degradation at free ends of plasmids (33). This expression of I–SceI enhanced homologous recombination might account for a reduced frequency of REMI in our several thousand-fold between repeats at integrated defective experiments. The degradation of ends should, however, affect copies of the neomycin gene when the I–SceI site was present both spontaneous illegitimate integration and REMI to the in one of the repeats (32). Since BglII and EcoRI have sites in same extent. Since we found that REMI was completely the target, but those sites have been removed from the targeting abolished, and spontaneous illegitimate integration only vector, we expected that the frequency of homologous integra- decreased to ∼60% (Table 2), Ku is specifically implicated in tion would be enhanced. On the contrary, we found that the DSB-induced illegitimate integration. For spontaneous inte- frequency of homologous integration was 2-fold reduced after gration events, an alternative pathway, however, may exist in co-transformation of restriction enzymes. agreement with the data of Liang et al. (32). Other studies The result that the frequency of homologous recombination report a defect in plasmid integration in the xrs6 mutant, which was decreased 2-fold after co-transformation with restriction is dependent on the DNA concentration. At low concentration, enzymes is only possible if 100% of the cells that received the very little or no difference was observed between mutant and transforming plasmid also received the restriction enzyme. If wild-type cells, but at high plasmid concentration a 5–10-fold some cells receive the plasmid alone, without the restriction difference was found (34). In that study, the polybrene and enzyme, this decrease is an underestimate of the true effect. calcium phosphate methods were used, which are quite The degree of this underestimate depends on the number of different from the electroporation method that we used, which cells co-transfected. For instance, if 50% of the cells received makes a comparison of the DNA concentrations difficult. the plasmid alone the frequency of gene targeting needs to be 4832 Nucleic Acids Research, 2001, Vol. 29, No. 23 completely abolished in the remaining 50% of the cells that REFERENCES were co-transfected with both the restriction enzyme and the 1. Schiestl,R.H. and Petes,T.D. (1991) Integration of DNA fragments by plasmid to result in an ∼2-fold overall decrease. illegitimate recombination in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA, 88, 7585–7589. Most previous experiments suggest that the illegitimate end- 2. Manivasakam,P. and Schiestl,R.H. 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Nucleic Acids ResearchOxford University Press

Published: Dec 1, 2001

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